A nested multiplex polymerase chain reaction assay for the differential identification of three zooanthroponotic chlamydial strains in porcine swab samples

Abstract

Porcine chlamydial infection is an enzootic infectious disease caused by multiple members of the family Chlamydiaceae (e.g. Chlamydophila abortus, Chlamydia suis, and Chlamydophila pneumoniae). Rapid and accurate differentiation of these pathogens is critical in the control and prevention of disease. The aim of the current study was to develop a nested multiplex polymerase chain reaction (nmPCR) assay to simultaneously detect the 3 chlamydial pathogens in clinical samples. In the first round of the nmPCR, 1 pair of family-specific primers were used to amplify the 1,100 base pair (bp) fragment of chlamydial ompA gene. In the second round of the nmPCR, 4 inner primers were designed for Ch. abortus, C. suis, and Ch. pneumoniae. Each pathogen produced a speciﬁc amplicon with a size of 340 bp, 526 bp, and 267 bp respectively. The assay was sensitive and speciﬁc for detecting target pathogens in both cell cultures and clinical specimens. The results, incorporated with the improved rapid DNA extraction protocol, suggest that the nmPCR could be a promising assay for differential identification of different chlamydial strains in pigs.

Keywords

nested multiplex polymerase chain reaction porcine

Introduction

Chlamydial infection is considered as an enzootic infectious disease of pigs.^4,16 Concurrent infection of chlamydial pathogens with other pig diseases generally causes wasting, respiratory signs, diarrhea, enlargement of lymph nodes, and increased mortality.³¹ The causative agents of porcine chlamydial disease include Chlamydophila abortus, Chlamydia suis, and Chlamydophila pneumoniae.^5,33 Chlamydophila abortus has not only been associated with enzootic abortion and other clinical signs in sheep and pigs,^21,26 but has also been related to cases of human disease.²² Chlamydia suis, which is a close relative of Chlamydia trachomatis, has been found in semen and genital tracts of boars, sows, bulls, and bucks; the clinical signs of the infected animals varied from conjunctivitis, to enteritis, to pneumonia.^14,25 Although there is no evidence about the possibility of infection in human beings with C. suis, the emergence of removable tetracycline-resistant plasmids in C. suis suggests a potential threat for the food safety of human beings.³⁴ Although Ch. pneumoniae is considered a human infectious agent, evidence indicates that a wide range of hosts for Ch. pneumoniae, including human beings, koalas, horses, reptiles, and pigs, exists and that a possible reason for such a wide range of hosts may be that Ch. pneumoniae potentially crosses the various host barriers through the processes of gene decay and plasmid loss in such hosts.^3,23 The high prevalence of Ch. pneumoniae–specific antibodies (22.5%) in the sera of boars from breeding stations also seemed to be a potential health hazard for human beings.⁵

Chlamydial infections can be detected either by isolation of pathogens or by screening for antibodies in tissue scrapings and smears, tissue sections, serum, and bodily secretions.^27,29 Because of the insufficient sensitivity, low specificity, and low pathogen loads of clinical samples, it is difficult to detect infection status by means of such tests.²⁹ Alternative methodologies based on nucleic acid amplification, such as polymerase chain reaction (PCR), which possess both unsurpassed sensitivity and specificity, have been recently used to diagnose human chlamydiosis.^2,24,30 Although real-time PCR assay and microarray-based methods are superior to traditional PCR assays in their sensitivity as well as throughput, the traditional PCR assays are easier to perform and standardize in different facilities.^28,29 The traditional PCR assays, which involve tedious DNA extraction procedures or combining a few pair of primers to detect 1 or very few targets in 1 reaction, are not convenient enough for the rapid identification of porcine chlamydial infection.⁷ For example, the successful detection of all related Chlamydophila and Chlamydia spp. of veterinary importance needs 6 separate, species-specific real-time PCR assays.²⁰ For the diagnosis of enzootic abortion, a multiplex PCR for the differential detection of Ch. abortus and Chlamydophila pecorum and a duplex real-time PCR for the specific detection of Chlamydophila psittaci and Ch. abortus have been developed.¹⁹ To the authors’ knowledge, a PCR assay for the simultaneous detection of the Ch. abortus, C. suis, and Ch. pneumoniae has not been applied. In the current study, a nested multiplex polymerase chain reaction (nmPCR) assay was developed to simultaneously detect such zooanthroponotic chlamydial pathogens from clinical samples.

Materials and methods

Reagents, cell, organisms, and their propagation

The following reagents were used: Eagle minimal essential medium (EMEM),^a fetal calf serum (FBS),^b genomic DNA (gDNA) isolation kit,^c PCR reagents (including 10× PCR buffer, deoxyribonucleotide triphosphate [dNTP] mix, and Taq polymerase),^d fluorescein isothiocyanate (FITC)-conjugated Chlamydiaceae-specific antibody,^e and antibiotics (gentamicin, vancomycin, cycloheximide).^f Chlamydophila abortus, C. suis, Ch. pneumoniae, and C. trachomatis were cultured in Henrietta Lacks (HeLa) 229 cell line as described in the World Organization for Animal Health (OIE) manual, with the following modifications.³⁵ Briefly, 100 µl of stock cultures were inoculated onto 24-well plates of exponentially growing HeLa 229 cell monolayers in EMEM supplemented with 5% FBS and antibiotics (gentamicin and vancomycin), and then centrifuged at 500 × g for 60 min at room temperature. The cultures were incubated for 3–5 days in a CO₂ incubator following addition of 0.5 µg/ml of cycloheximide. The supernatant of the cell culture was harvested and mixed with 2× sucrose–phosphate–glutamate (SPG) buffer (149.2 g/l of sucrose, 1.02 g/l of monopotassium phosphate, 2.474 g/l of dipotassium phosphate, 1.442 g/l of L-glutamic acid). The elementary body contained in the supernatant was titrated by immunofluorescent staining with an FITC-conjugated Chlamydiaceae-specific antibody. The number of inclusions was counted in 20 random fields and calculated as inclusion-forming units (IFU) per milliliter. The supernatant of the cell culture was used for the PCR assay. Other organisms (see Table 1), including Pasteurella multocida, Legionella pneumophila, Mycoplasma hyopneumoniae, Brucella abortus, Listeria monocytogenes, Actinobacillus pleuropneumoniae, enterotoxigenic Escherichia coli, Haemophilus parasuis, and Klebsiella pneumoniae, were cultured in proper medium and used for DNA extraction.

Table 1.

Chlamydial strains used in the current study for the development of the nested multiplex polymerase chain reaction assay.*

Name	Source
Actinobacillus pleuropneumoniae	Swine isolate
Brucella abortus	Cow isolate
Chlamydia suis	ATCC VR1474
Chlamydia trachomatis	CVCC 1950
Chlamydophila abortus †	NATSEL 0624
Chlamydophila abortus ‡	Jizhang Zhou
Chlamydophila pneumoniae	ATCC 53592
Enterotoxigenic Escherichia coli	ATCC 8739
Haemophilus parasuis	Swine isolate
Klebsiella pneumoniae	ATCC 700603
Legionella pneumophila	ATCC 33152
Listeria monocytogenes	ATCC 7644
Mycoplasma hyopneumoniae	Swine isolate
Pasteurella multocida	Swine isolate

ATCC = American Type Culture Collection, Manassas, Virginia; CVCC = China Veterinary Culture Collection Center, Beijing, China; NATSEL = National Animal TSE Lab, China Agriculture University, Beijing, China.

†

Strain was isolated from poultry. The ompA gene was sequenced in the current study (data not shown).

‡

Strain was isolated from pig and provided by Dr. Jizhang Zhou (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China). The ompA gene was sequenced in the current study (data not shown).

DNA extraction and optimized protocol for swabs

The DNA was extracted from the organism cultures and clinical samples by using 2 different protocols. In protocol 1, DNA was extracted by using a silicon column–based gDNA isolation kit,^c according to the manufacturer’s instructions. In protocol 2, samples were centrifuged at 500 × g for 5 min at room temperature, and the supernatant was collected. After centrifuging the supernatant at 10,000 × g for 30 min, the sediment was resuspended in 11 µl of Mini-Q water and the mixture boiled at 100°C for 15 min. Then, the mixture was centrifuged at 500 × g for 1 min, and all of the supernatant was used for the PCR assay.

To evaluate the reliability of protocol 2 for DNA extraction from swabs, approximately 10 ml of supernatants from chlamydia-free swabs were pooled by centrifuging at 500 × g for 5 min and stored at 4°C until use. A cell culture containing Ch. abortus (5 × 10² IFU/ml), C. suis (3 × 10² IFU/ml), and Ch. pneumoniae (2 × 10² IFU/ml) was mixed in equal volumes and serially diluted to 10⁻⁴ with the pooled supernatants. Using protocols 1 and 2 separately, the DNA was extracted from 300 µl of supernatant obtained at dilution, and then used for PCR assays.

Primer design

A multi-alignment–based criterion was used for primer design.¹³ Primers were designed in the domains that were conserved among family or species, forming a nested strategy (Table 2). The primers were evaluated with the National Center for Biotechnology Information Basic Local Alignment Search Tool (BLAST) for their specificity, then synthesized.^g

Table 2.

Primers used in the current study to amplify the chlamydial strains.*

Pathogens	ompA gene position†	Primer sequence (5′-3′)
Chlamydiaceae	86 → 106	H_up primer: 5′-CTCCTTRCAAGCYYTGCCTGT-3′
	1232 ← 1206	H_low primer: 5′-GTGAGCWGCTCTTTCRTYRATTAARCG-3′ (1,100 bp)
	193 → 212	H_up-n primer: 5′-TACGGAGAYTATGTTTTYGA-3′
Chlamydophila abortus	518 ← 534	S_low primer: 5′-GCCTTGAGTGATGCCTA-3′ (340 bp)
Chlamydophila pneumoniae	443 ← 462	C_low primer: 5′-GAGATTGAACGCTGTAGAGT-3′ (267 bp)
Chlamydia suis	678 ← 697	F_low primer: 5′-GAGGTTTGTTGATGGTGAAT-3′ (526 bp)

R = A/G; Y = C/T; W = A/T; bp = base pair. Numbers in parentheses are length of product.

†

“→” indicates the direction is from upstream to downstream on the sense strand of the genomic DNA; “←” indicates the reverse direction.

Nested multiplex polymerase chain reaction assay

Polymerase chain reaction assay mixtures were prepared in a PCR workstation, and the amplification and analysis of PCR products were performed in separate locations. The PCR assay was optimized by varying primer concentrations and by methodical variation of each test parameter under standard PCR conditions. During the first round of the PCR assay, samples were amplified in a 25-µl reaction containing 1× PCR buffer, 200 µmol/l of dNTP mix, 1.5 mmol/l of MgCl₂, 1.25 U of Taq polymerase, 0.3 µmol/l of H_up primer, 0.3 µmol/l of H_low primer and 50–100 ng of DNA as the template. The thermal profile consisted of 1 cycle at 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 59°C for 35 sec, and 72°C for 1 min, and 1 cycle of final extension at 72°C for 8 min. In the second round of the PCR assay, samples were amplified in a 25-µl reaction containing 1× PCR buffer, 200 µmol/l of dNTP mix, 2.0 mmol/l of MgCl₂, 1 U of Taq polymerase, 0.4 µmol/l of H_up-n primer, 0.3 µmol/l of S_low primer, 0.2 µmol/l of C_low primer, 0.1 µmol/l of F_low primer, and 0.5–1.0 µl of product of the first round of nmPCR assay as the template. The thermal profile consisted of 1 cycle at 95°C for 5 min followed by 30 cycles at 95°C for 30 sec, 52°C for 30 sec, and 72°C for 30 sec, and 1 cycle of final extension at 72°C for 8 min.

The PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualized under ultraviolet light. The product size of the first amplification round was 1,100 bp. The specific product size of the second round nmPCR assay was 340 bp (Ch. abortus), 526 bp (C. suis), and 267 bp (Ch. pneumoniae).

The evaluation of specificity and sensitivity

The DNA used in the specificity assay was extracted from a panel of pathogens listed in Table 1. First, the DNA was amplified by the first round nmPCR. Then, 1 µl of product from the first round PCR was amplified by the second round species-specific nmPCR. To assess the sensitivity, the supernatant of cell culture containing Ch. abortus (5 × 10³ IFU/ml), C. suis (3 × 10³ IFU/ml), and Ch. pneumoniae (2 × 10³ IFU/ml) was mixed in equal volume and serially diluted to 10⁻⁴ with 0.1 mol/l of phosphate buffered saline. Then, using protocol 2, 300 µl of supernatant from each dilution was extracted, and DNA was amplified by nmPCR assay. Equal amounts of DNA were also amplified by the 16S ribosomal DNA (rDNA) nmPCR assay¹⁸ and the 23S rDNA real-time PCR assay.¹¹

Evaluation of the nested multiplex polymerase chain reaction assay with clinical samples

Swabs (vaginal, nasopharyngeal, and rectal) were collected from 122 adult sows from 2 local breeding farms diagnosed with a high prevalence of anti-Chlamydiaceae antibody and subclinical reproductive and respiratory disease. Swabs were freshly soaked into 1× SPG buffer (supplemented with 10% fetal bovine serum, 500 µg/ml of streptomycin, 50 µg/ml of gentamicin, and 50 µg/ml of amphotericin B) and kept at 4°C. According to protocol 2, 300 µl of supernatant was used to extract DNA, and the DNA templates were detected by both nmPCR and 23S rDNA real-time PCR assays. Both negative (water) and positive controls (plasmid) were also included in the PCR assays.

Pathogen isolation and sequencing

Three hundred microliters of the swab supernatant was used for pathogen isolation, which was performed according to the OIE manual by inoculating the yolk sac of 6–7-day-old embryonated chicken eggs. After 7 days, the yolk sac and allantoic fluid were collected. Pathogens were examined by nmPCR assay. The negative samples were treated as described earlier and inoculated into chicken egg embryos for another passage.²⁰ The positive PCR products of the nmPCR assay were cloned into pMD19-T vector. Positive clones were sequenced and multi-aligned with the appropriate reference sequences (C. suis AF269274; Ch. pneumoniae M69230.1; Ch. abortus AF269256). The sequences were analyzed using Molecular Evolutionary Genetics Analysis (MEGA 4.0) software.³²

Results

Sequence analysis and primer design

A phylogenetic tree was used for the primer design by multi-alignment of sequences, which were grouped into 9 separate branches, with patterns identical to the 16S rDNA phylogenetic tree built by the international classification organization.¹⁰ One pair of family-specific primers was designed on the family-specific domain and named H_up and H_low. Another forward family-specific sense primer was designed 200 bp downstream of the primer H_up and named H_up-n. Three antisense primers specific for Ch. abortus, C. suis, and Ch. pneumoniae were individually designed on the species-specific domains that were located between H_up and H_low primers. These primers formed the nested pattern (Fig. 1).

Figure 1.

Primers used in the current study were spanning the ompA gene. Primers H_up, H_low, and H_up-n were chlamydial family-specific. Primers S_low, C_low, and F_low were specific for Chlamydophila abortus, Chlamydophila pneumoniae, and Chlamydia suis, respectively.

Specificity of the nested multiplex polymerase chain reaction assays

The 1,100-bp family-specific fragment was amplified from Ch. abortus, C. suis, Ch. pneumonia, and C. trachomatis in the first round of nmPCR. There was no amplification from the other microorganisms (Fig. 2A). A second round of nmPCR further analyzed the products of the first round amplification. As shown in Figure 2B, fragments specific to Ch. abortus, C. suis, and Ch. pneumoniae were successfully amplified, respectively; however, C. trachomatis and other microorganisms could not yield specific amplicons. The DNA of Ch. abortus, C. suis, and Ch. pneumoniae were examined by PCR assays separately and as mixtures. The PCR assay could simultaneously detect these 3 species in every arrangement as shown in Figure 3.

Figure 2.

Results of the specificity test of the nested multiplex polymerase chain reaction (nmPCR) assay. A, the first round of nmPCR. B, the second round of nmPCR. Lane M: DL2000 DNA marker.

Figure 3.

Simultaneous detection of multiple chlamydial organisms in 1 reaction. Lane 1: Chlamydophila abortus⁺ , Chlamydia suis⁺ , and Chlamydophila pneumonia⁺ ; lane M: DL2000 DNA marker; lane 2: Ch. pneumoniae⁺ ; lane 3: Ch. abortus⁺ ; lane 4: C. suis⁺ ; lane 5: Ch. pneumoniae ⁺ and Ch. abortus⁺ ; lane 6: Ch. pneumoniae⁺ and C. suis⁺ ; lane 7: Ch. abortus⁺ and C. suis⁺ .

Sensitivity of the nested multiplex polymerase chain reaction assays

As shown in Figure 4, nmPCR assay could simultaneously detect Ch. abortus, C. suis, and Ch. pneumoniae at dilutions as low as 10⁻³. The sensitivity of the 2-step nmPCR assay developed was under 5 IFU/ml, and it was 10 times more sensitive than that of the 16S rDNA nPCR and 23S rDNA real-time PCR arrays as shown in Figure 5.

Figure 4.

Comparison of the analytical sensitivity between 2 nested multiplex polymerase chain reaction assays.

Figure 5.

Results of the sensitivity test of the 23S real-time polymerase chain reaction (PCR) assay. The serially diluted chlamydial organisms were plotted with relative fluorescence intensity. Relative fluorescence intensity was calculated by: (100 – threshold Ct) × 100%, where Ct indicates the threshold cycle numbers in real-time PCR. The relative fluorescence intensity of the negative samples was considered as zero.

Optimized protocol for swab DNA extraction

The 2 protocols could extract sufficient DNA for successful amplification of specific fragments of Ch. abortus, C. suis, and Ch. pneumoniae at the 10⁻¹dilution. At the dilution of 10⁻², the nmPCR of DNA extracted by protocol 1 (commercial DNA kit) could not amplify visible fragments. However, there were visible amplifications of all the 3 targets by protocol 2 (Fig. 6). Thus, protocol 2 was 10 times more efficient than protocol 1 for DNA extraction.

Figure 6.

Optimized protocols used for the DNA extraction of swabs to detect Chlamydophila abortus, Chlamydia suis, and Chlamydophila pneumonia.

Evaluation of the nested multiplex polymerase chain reaction assay with clinical samples

A different prevalence of chlamydial infection was observed in swabs by different methods (Table 3). The positive rate of the nmPCR assay is 9% in vaginal swabs, 3% and 5% higher than that of 23S real-time PCR and pathogen isolation, respectively. The nmPCR assay gave rise to 22% positive rate in nasopharyngeal swabs, which is 2% higher than that of 23S real-time PCR. While the positive rate resulted by the nmPCR assay is 4% in rectal swabs, it was negative from pathogen isolation and 23S real-time PCR. The infection type of the sow detected by nmPCR assay was shown in Table 4. Chlamydophila abortus was only detected from vaginal swabs. Six vaginal swabs and 3 rectal swabs were positive for C. suis. Meanwhile, Ch. pneumoniae was found in nasopharyngeal and rectal swabs. However, the 23S real-time PCR could not differentiate among them.

Table 3.

Number of positive samples obtained with nested multiplex polymerase chain reaction (nmPCR), 23S real-time PCR, and pathogen isolation.

Source	nmPCR	23S real-time PCR	Pathogen isolation
Vaginal swabs	11	7	5*
Nasopharyngeal swabs	27	25	0
Rectal swabs	5	0	0

Five isolates were confirmed as Chlamydophila abortus using nmPCR.

Table 4.

Number of positive samples detected by nested multiplex polymerase chain reaction assay.

Source	Chlamydophila abortus	Chlamydia suis	Chlamydophila pneumoniae
Vagina swabs	5	6	0
Nasopharyngeal swabs	0	0	27
Rectal swabs	0	3	2

Positive products of the second round nmPCR were cloned into pMD19-T vector, and 16 clones were successfully sequenced. The multi-alignment shown illustrates that all C. suis clones were obviously different from the reference strain (C. suis S45 strain) maintained in the authors’ laboratory (Fig. 7). The sequences of Ch. pneumoniae and Ch. abortus acquired from nmPCR products were approximately the same as the reference strains.

Figure 7.

Multi-alignment of the sequences of Chlamydia suis isolates and reference strain (C. suis S45 strain, AF269274). Compared to the S45 strain, the isolates contained triple-base deletion (609–611 bp) and other minor differences.

Discussion

Primer design is the most critical part of PCR-based diagnostic techniques.⁸ The nested PCR strategy shows a significant advantage in both sensitivity and specificity.^1,17 The specificity of the nmPCR assay was confirmed by the evaluation of 14 bacterial pathogens in the test of specificity. The nmPCR assay possessed good specificity even in the detection of multiple targets. In the nmPCR, the concentration of magnesium, primers, and dNTP were optimized followed by a step-by-step protocol previously described.¹⁵ To minimize false-negative results, the amplification efficiency of the nmPCR assay was optimized to be individually equivalent for each of Ch. abortus, C. suis, and Ch. pneumoniae. Thus, the nmPCR assay could not miss the target due to the efficiency bias. To evaluate the sensitivity of the nmPCR assay, 2 other PCR assays were used to detect the serially diluted live organism. Owing to the nested amplification of the targets, the sensitivity of the nmPCR assay was greatly elevated.

Swabbing is a harmless and efficient sampling method in chlamydial diagnosis.¹² However, it was noted that inhibitors contained in swabs might generally decrease the activity of DNA polymerase enzyme.⁶ Therefore, the direct extraction method (protocol 2) was 10 times more efficient than the silicon column–based method (protocol 1). Such observation might be explained by the relative long time centrifugation, which can gather more chlamydial agents, thus avoiding the loss of DNA as in protocol 1.⁹ Compared with the traditional methods, protocol 2 was simpler and easier to perform at a significantly lower cost.

In pathogen isolation, Ch. abortus was positive only in 5 cases out of 122 swabs, which was identical to the results of the nmPCR assay. The 23S real time PCR had a lower increase of positive rates compared with nmPCR. This is likely due to the potential inhibitors in the swabs, while nmPCR could avoid the inhibition because of its 2 nested rounds of PCR reaction. The results of the nmPCR assay corresponded with the chlamydial antibody level and clinical status. It is necessary to further evaluate the potential hazard for workers in the pig farming industry from Ch. pneumoniae emerging in pig breeding farms.⁵

The reliability of the nmPCR assay was confirmed by sequencing the products. The reason for this phenomenon may be conservative ompA gene sequence among short PCR products. In summary, the nmPCR assay developed in the current study offered a sensitive and specific screening method for the simultaneous detection of Ch. pneumoniae, Ch. abortus, and C. suis in pigs.

Footnotes

Acknowledgements

The authors thank Dr. John Yang (Lincoln University, Jefferson City, Missouri) and Miss Rasha Khalil for revising of the manuscript, as well as Dr. Jizhang Zhou (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China) for kindly providing the DNA of Ch. abortus strain. Yingguo Li and Yu Wang contributed equally to the work.

a.

American Type Culture Collection, Manassas, VA.

b.

HyClone laboratories Inc., Victoria, Australia.

c.

Omega Bio-Tek Inc., Norcross, GA.

d.

Promega Corp., Shanghai, China.

e.

Abcam Corp., Cambridge, United Kingdom.

f.

Sangon Biotech Corp., Shanghai, China.

g.

Takara Corp., Da liang, China.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants (CSTC 2009CB1012, CSTC 2007AC1002, CSTC 2009BB1181) from the Committee of Science and Technology of Chongqing.

References

Apfalter

Hammerschlag

Boman

: 2003, Reliability of nested PCR for the detection of Chlamydia pneumoniae in carotid artery atherosclerosis. Stroke 34:e73–e75.

Berri

Rekiki

Boumedine

Rodolakis

: 2009, Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant’s clinical samples using multiplex PCR. BMC Microbiol 9:130.

Bodetti

Jacobson

Wan

.: 2002, Molecular evidence to support the expansion of the host range of Chlamydophila pneumoniae to include reptiles as well as humans, horses, koalas and amphibians. Syst Appl Microbiol 25:146–152.

Busch

Thoma

Schiller

.: 2000, Occurrence of chlamydiae in the genital tracts of sows at slaughter and their possible significance for reproductive failure. J Vet Med B Infect Dis Vet Public Health 47:471–480.

Canderle

Pospisil

Stroblova

: 2005, Chlamydophila pneumoniae antibodies in swine. Acta Vet Brno 74:81–86.

Costa

Fairley

Garland

Tabrizi

: 2009, Evaluation of self-collected urine dip swab method for detection of Chlamydia trachomatis . Sex Health 6:213–216.

DeGraves

Gao

Kaltenboeck

: 2003, High-sensitivity quantitative PCR platform. Biotechniques 34:106–110, 112–105.

Dieffenbach

Lowe

Dveksler

: 1993, General concepts for PCR primer design. PCR Methods Appl 3:S30–37.

Echavarria

Sanchez

Kolavic-Gray

.: 2003, Rapid detection of adenovirus in throat swab specimens by PCR during respiratory disease outbreaks among military recruits. J Clin Microbiol 41:810–812.

10.

Everett

Bush

Andersen

: 1999, Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 49(pt 2):415–440.

11.

Everett

Hornung

Andersen

: 1999, Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests. J Clin Microbiol 37:575–580.

12.

Fang

Husman

DeSilva

.: 2008, Evaluation of self-collected vaginal swab, first void urine, and endocervical swab specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in adolescent females. J Pediatr Adolesc Gynecol 21:355–360.

13.

Frech

Breuer

Ronacher

.: 2009, Hybseek: pathogen primer design tool for diagnostic multi-analyte assays. Comput Methods Programs Biomed 94:152–160.

14.

Guscetti

Schiller

Sydler

.: 2009, Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45. Vet Microbiol 135:157–168.

15.

Henegariu

Heerema

Dlouhy

.: 1997, Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23:504–511.

16.

Kauffold

Melzer

Berndt

.: 2006, Chlamydiae in oviducts and uteri of repeat breeder pigs. Theriogenology 66:1816–1823.

17.

Lan

Ossewaarde

Walboomers

.: 1994, Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR. J Clin Microbiol 32:528–530.

18.

Messmer

Skelton

Moroney

.: 1997, Application of a nested, multiplex PCR to psittacosis outbreaks. J Clin Microbiol 35:2043–2046.

19.

Pantchev

Sting

Bauerfeind

.: 2009, New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples. Vet J 181:145–150.

20.

Pantchev

Sting

Bauerfeind

.: 2010, Detection of all Chlamydophila and Chlamydia spp. of veterinary interest using species-specific real-time PCR assays. Comp Immunol Microbiol Infect Dis 33:473–484.

21.

Polkinghorne

Borel

Becker

.: 2009, Molecular evidence for chlamydial infections in the eyes of sheep. Vet Microbiol 135:142–146.

22.

Pospischil

Thoma

Hilbe

.: 2002, Abortion in woman caused by caprine Chlamydophila abortus (Chlamydia psittaci serovar 1). Swiss Med Wkly 132:64–66.

23.

Pospisil

Canderle

: 2004, Chlamydia (Chlamydophila) pneumoniae in animals: a review. Vet Med Czech 49:129–134.

24.

Raherison

Clerc

Trombert

.: 2009, Real-time high resolution melting PCR for identification of the Swedish variant of Chlamydia trachomatis . J Microbiol Methods 78:101–103.

25.

Rogers

Andersen

: 2000, Intestinal lesions caused by a strain of Chlamydia suis in weanling pigs infected at 21 days of age. J Vet Diagn Invest 12:233–239.

26.

Sachse

Grossmann

: 2002, Chlamydial diseases of domestic animals—zoonotic potential of the agents and diagnostic issues. Dtsch Tierarztl Wochenschr 109:142–148.

27.

Sachse

Grossmann

Jager

.: 2003, Detection of Chlamydia suis from clinical specimens: comparison of PCR, antigen ELISA, and culture. J Microbiol Methods 54:233–238.

28.

Sachse

Hotzel

Slickers

.: 2005, DNA microarray-based detection and identification of Chlamydia and Chlamydophila spp. Mol Cell Probes 19:41–50.

29.

Sachse

Vretou

Livingstone

.: 2009, Recent developments in the laboratory diagnosis of chlamydial infections. Vet Microbiol 135:2–21.

30.

Schaeffer

Henrich

: 2008, Rapid detection of Chlamydia trachomatis and typing of the Lymphogranuloma venereum associated L-serovars by TaqMan PCR. BMC Infect Dis 8:56.

31.

Schautteet

Beeckman

Delava

Vanrompay

: 2010, Possible pathogenic interplay between Chlamydia suis, Chlamydophila abortus and PCV-2 on a pig production farm. Vet Rec 166:329–333.

32.

Tamura

Dudley

Nei

Kumar

: 2007, MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599.

33.

Teankum

Pospischil

Janett

.: 2006, Detection of chlamydiae in boar semen and genital tracts. Vet Microbiol 116:149–157.

34.

Teankum

Pospischil

Janett

.: 2007, Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks. Theriogenology 67:303–310.

35.

World Organization for Animal Health (OIE): 2008, Manual of diagnostic tests and vaccines for terrestrial animals, pp. 431–442. OIE, Paris, France.